The Biochemistry and Physiology of A Disintegrin and Metalloproteinases (ADAMs and ADAM-TSs) in Human Pathologies.

Metalloproteinases are a group of proteinases that plays a substantial role in extracellular matrix remodeling and its molecular signaling. Among these metalloproteinases, ADAMs (a disintegrin and metalloproteinases) and ADAM-TSs (ADAMs with thrombospondin domains) have emerged as highly efficient contributors mediating proteolytic processing of various signaling molecules. ADAMs are transmembrane metalloenzymes that facilitate the extracellular domain shedding of membrane-anchored proteins, cytokines, growth factors, ligands, and their receptors and therefore modulate their biological functions. ADAM-TSs are secretory, and soluble extracellular proteinases that mediate the cleavage of non-fibrillar extracellular matrix proteins. ADAMs and ADAM-TSs possess pro-domain, metalloproteinase, disintegrin, and cysteine-rich domains in common, but ADAM-TSs have characteristic thrombospondin motifs instead of the transmembrane domain. Most ADAMs and ADAM-TSs are activated by cleavage of pro-domain via pro-protein convertases at their N-terminus, hence directing them to various signaling pathways. In this article, we are discussing not only the structure and regulation of ADAMs and ADAM-TSs, but also the importance of these metalloproteinases in various human pathophysiological conditions like cardiovascular diseases, colorectal cancer, autoinflammatory diseases (sepsis/rheumatoid arthritis), Alzheimer's disease, proliferative retinopathies, and infectious diseases. Therefore, based on the emerging role of ADAMs and ADAM-TSs in various human pathologies, as summarized in this review, these metalloproteases can be considered as critical therapeutic targets and diagnostic biomarkers.

Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura.

Antibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G-type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

The evolution of ADAM gene family in eukaryotes.

The ADAM (A Disintegrin And Metalloprotease) gene family encodes proteins with adhesion and proteolytic functions. ADAM proteins are associated with diseases like cancers. Twenty ADAM genes have been identified in humans. However, little is known about the evolution of the family. We analyzed the repertoire of ADAM genes in a vast number of eukaryotic genomes to clarify the main gene copy number expansions. For the first time, we provide compelling evidence that early-branching green algae (Mamiellophyceae) have ADAM genes, suggesting that they originated in the last common ancestor of eukaryotes, before the split of plants, fungi and animals. The ADAM family expanded in early metazoans, with the most significative gene expansion happening during the first steps of vertebrate evolution. We concluded that most of mammal ADAM diversity can be explained by gene duplications in early bone fish. Our data suggest that ADAM genes were lost early in green plant evolution.

Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Meprin ß is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin ß, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin ß substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin ß and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin ß in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin ß caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin ß and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin ß and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin ß/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin ß with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Functions of 'A disintegrin and metalloproteases (ADAMs)' in the mammalian nervous system.

'A disintegrin and metalloproteases' (ADAMs) are a family of transmembrane proteins with diverse functions in multicellular organisms. About half of the ADAMs are active metalloproteases and cleave numerous cell surface proteins, including growth factors, receptors, cytokines and cell adhesion proteins. The other ADAMs have no catalytic activity and function as adhesion proteins or receptors. Some ADAMs are ubiquitously expressed, others are expressed tissue specifically. This review highlights functions of ADAMs in the mammalian nervous system, including their links to diseases. The non-proteolytic ADAM11, ADAM22 and ADAM23 have key functions in neural development, myelination and synaptic transmission and are linked to epilepsy. Among the proteolytic ADAMs, ADAM10 is the best characterized one due to its substrates Notch and amyloid precursor protein, where cleavage is required for nervous system development or linked to Alzheimer's disease (AD), respectively. Recent work demonstrates that ADAM10 has additional substrates and functions in the nervous system and its substrate selectivity may be regulated by tetraspanins. New roles for other proteolytic ADAMs in the nervous system are also emerging. For example, ADAM8 and ADAM17 are involved in neuroinflammation. ADAM17 additionally regulates neurite outgrowth and myelination and its activity is controlled by iRhoms. ADAM19 and ADAM21 function in regenerative processes upon neuronal injury. Several ADAMs, including ADAM9, ADAM10, ADAM15 and ADAM30, are potential drug targets for AD. Taken together, this review summarizes recent progress concerning substrates and functions of ADAMs in the nervous system and their use as drug targets for neurological and psychiatric diseases.

An Overview of ADAM9: Structure, Activation, and Regulation in Human Diseases.

ADAM9 (A disintegrin and a metalloprotease 9) is a membrane-anchored protein that participates in a variety of physiological functions, primarily through the disintegrin domain for adhesion and the metalloprotease domain for ectodomain shedding of a wide variety of cell surface proteins. ADAM9 influences the developmental process, inflammation, and degenerative diseases. Recently, increasing evidence has shown that ADAM9 plays an important role in tumor biology. Overexpression of ADAM9 has been found in several cancer types and is correlated with tumor aggressiveness and poor prognosis. In addition, through either proteolytic or non-proteolytic pathways, ADAM9 promotes tumor progression, therapeutic resistance, and metastasis of cancers. Therefore, comprehensively understanding the mechanism of ADAM9 is crucial for the development of therapeutic anti-cancer strategies. In this review, we summarize the current understanding of ADAM9 in biological function, pathophysiological diseases, and various cancers. Recent advances in therapeutic strategies using ADAM9-related pathways are presented as well.

Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors.

Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE.

Differences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin α, Meprin ß and MT1-MMP.

Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin ß, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes.

The soluble protease ADAMDEC1 released from activated platelets hydrolyzes platelet membrane pro-epidermal growth factor (EGF) to active high-molecular-weight EGF.

Platelets are the sole source of EGF in circulation, yet how EGF is stored or released from stimulated cells is undefined. In fact, we found platelets did not store EGF, synthesized as a single 6-kDa domain in pro-EGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released high-molecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and the transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released soluble activity that cleaved this pro-EGF1020-1027 peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE (tumor necrosis factor-α-converting enzyme) inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed pro-EGF1020-1027, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells and EGFR-dependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF Arg1023 to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF1020-1027 peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF to soluble HMW-EGF, that HMW-EGF is active, that proteolytic cleavage of pro-EGF first occurs at the C-terminal arginyl residue of the EGF domain, and that proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity from activated platelets.

CD4+ T cells from patients with acquired thrombotic thrombocytopenic purpura recognize CUB2 domain-derived peptides.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11- and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR- or ASYILIRD-reactive CD4(+)T cells are present in peripheral blood mononuclear cells from HLA-DRB1*11 and HLA-DRB1*03-positive subjects with acquired TTP. The presence of ADAMTS13-reactive CD4(+)T cells was addressed by flow cytometry and the expression of activation marker CD40 ligand by CD4(+)T cells. FINVAPHAR-reactive CD4(+)T cells were identified in an HLA-DRB1*11-positive patient during the acute phase of the disease whereas ASYILIRD-positive CD4(+)T cells were identified in a DRB1*03-positive patient with acquired TTP. Frequencies of CUB2 domain-reactive CD4(+)T cells ranged from 3.3% to 4.5%. Control peptides in which the anchor residues were modified did not induce activation of CD4(+)T cells. Taken together, our data provide evidence for the involvement of CUB2 domain-reactive CD4(+)T cells in the etiology of acquired TTP.

N-linked glycan stabilization of the VWF A2 domain.

Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+)binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca(2+)binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13.

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1' substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation.

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS.

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.

The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

Allosteric activation of ADAMTS13 by von Willebrand factor.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

Conformational activation of ADAMTS13.

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼ 2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼ 2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼ 2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.

Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10.

Intrinsically disordered protein regions are widely distributed in the cytoplasmic domains of many transmembrane receptors. The cytoplasmic domain of a disintegrin and metalloprotease (ADAM)10, a transmembrane metalloprotease mediating ectodomain shedding of diverse membrane proteins, was recently suggested to mediate the homodimerization of ADAM10. Here we show that a recombinant cytoplasmic domain of ADAM10 (A10Cp) is unstructured as judged by its susceptibility to limited trypsin digestion and its circular dichroism spectrum. In comparison, recombinant transmembrane-cytoplasmic domain of ADAM10 (A10TmCp) reconstituted in dodecylphosphocholine (DPC) micelles exhibits much greater resistance to trypsin digestion, with its cytoplasmic domain taking on a significant ordered structure. FRET analysis demonstrates that, although A10Cp remains monomeric, A10TmCp forms a tight homodimer (K(d) ∼ 7 nM) in DPC micelles. Phospholipid-conjugated A10Cp dose-dependently inhibits formation of A10TmCp homodimer, whereas A10Cp achieves only limited inhibition. Placing the transmembrane and cytoplasmic domains of ADAM10, but not the transmembrane domain alone, in their native orientation in the inner membrane of Escherichia coli produces specific and strong dimerization signal in the AraC-based transcriptional reporter assay. A chimeric construct containing the otherwise monomeric transmembrane domain of L-selectin and the cytoplasmic domain of ADAM10 produces a similar dimerization signal. Overall, these results demonstrate that a transmembrane domain imparts a stable structure to the adjacent and intrinsically disordered cytoplasmic domain of ADAM10 to form a homodimer in the membrane. This finding advances our understanding of the regulatory mechanism of ADAMs and has general implications for membrane-protein interactions in the process of transmembrane signaling.